Currently, scientific research is moving in three promising directions to elucidate the biomedical properties of human embryonic stem cells: a) identification of molecular events that play a key role in restricting pluripotency of embryonic stem cells, which would facilitate understanding of why pluripotency is lost in adult cells, as well as when, how, and why a stem cell differentiates into another type of cell; b) identification of an ultimate adult stem cell (body master cell), which can trans-dedifferentiate itself into virtually any of 200 types of cells in the human body; and c) identification of reprogramming factors which, under specific conditions, can orchestrate dedifferentiation of adult somatic cells into viable embryonic stem cells able to proliferate in an undifferentiated state while retaining pluripotency.
Recent studies have revealed some remarkable molecular events underlying the reprogramming of adult cells including: an activation of human Oct-4 gene, which serves as a convenient marker for nearby presence of reprogramming factors and maintains pluripotency of stem cells (Byrne J. A. et al., Current Biology, Jul. 15, 2003, 13(14):1206–1213); identification of germ cell nuclear factor (GCNF-receptor), which represses the Oct-4 gene, steadily decreasing its activity as embryonic stem cells differentiate and eventually restricts Oct-4's expression in the body's somatic cells, leaving expression only in the germ cell lineage (Schler H, Applied Genetics News, December, 2001); reversible disassembly of somatic nucleoli in Xenopus germ cells by the germ cell proteins FRGY2a and FRGY2b, which reversibly disassemble somatic nucleoli structure in ooplasm, independently of continuing ribosomal RNA transcription (Koichi Gonda et al., Nature Cell Biology, March, 2003, 5(3):205–210); and identification of a key molecule in this process: chromatin-remodeling nucleosomal adenosine triphosphate (ATPase) ISWI, which actively erases the TATA binding protein from association with the nuclear matrix which partially explains dramatic reshuffling of egg proteins when many of them are specifically lost from nuclei, and others are taken up from the frog's ooplasm (Nobuaki K. et al., Science, Sep. 29, 2000: 2360–2362).
Stem cell biology is currently facing a significant ethical dilemma associated with therapeutic cloning, which involves the derivation of stem cells from human embryos. In November of 2001, the privately funded U.S. based company Advanced Cell Technology Inc. announced that it had created a cloned human embryo using a nuclear transfer procedure. It was reported that the cloned embryo reached only the six-cell stage and failed to form a blastocyst, which is necessary for harvesting of human pluripotent embryonic stem cells (CNN/SCI-Tech, Nov. 26, 2001). Reprogramming of adult human cells into embryonic stem cells (ESC) is a way to bypass creation of embryos, but these experiments usually result in hybrids containing chromosomes from both the enucleated oocyte and the adult donor cell. Such genetic mismatch cannot be used in patients.
Reprogramming of two pre-fused ES cells carried out by a nuclei separation procedure utilizing a chemical inhibitor, which prevents the nuclei from joining together, is also an inconvenient and risky technique. Several research teams recently have attempted to dedifferentiate adult cells into primordial ES cells using an ooplasm “cocktail” made from frog eggs as a source of biochemical factors, which can be responsible for reprogramming of adult cells. These experiments are promising, but the “exact nature and number of the factors is unknown” (Dennis, C. Nature, Dec. 4, 2003, 426(6966):490–491).
The present inventor hypothesizes that separation of cytoplasmic and nuclear materials in both donor and recipient cells during the nuclear transfer procedure or during preparation of ooplasm is an unnecessary and undesirable intervention that can disrupt very important biochemical pathways that are naturally present and responsible for reprogramming of adult donor cells. Isolation from the endogenous biochemical network can lead to an incomplete and inaccurate picture of events involved in the dedifferentiation of adult cells. This is a major disadvantage that appears to be associated with inducing stem cells to proliferate in an undifferentiated state of pluripotency.
There is a need for a methodological approach that would allow scientists to directly reprogram adult cells, thereby bypassing the need for a nuclear transfer procedure, which requires dramatic intervention in chromosomal architecture in both donor and recipient cells.